Part:BBa_K4729707

Ptac + VirG TiBo542 N80D + repABCa

Introduction

Agrobacterium mediated transformation is one of the most prolific methods for plant engineering, by far the most common method for iGEM teams as well. However, working with Plant Synbio is still far from a straightforward endeavor. This year, we set our sights on creating molecular tools that facilitate achieving successful transformation in non-model plant species, one of them is our Best Composite Part.

In nature, members of the genus Agrobacterium (Alphaproteobacteria) are soil-borne plant pathogens that integrate hormone producing genes in the host plant genome to cause growth deformities and tumors. This mechanism can be explored by substituting the oncogenes by genes of interest, thus integrating them in the plant chromosomes. The most well known strain, Agrobacterium tumefaciens, promotes crown-gall disease in most dicotyledonous plants, however, other strains such as Agrobacterium rhizogenes are also able to transform plant cells (Bahramnejad et al., 2019^[2]; Barton et al., 2018^[3]). Despite their wide use, Agrobacterium mediated plant transformation is still only well established for a handful of model organisms, which makes it hard for iGEM teams to engineer local species.

Part of the problem lies in the high specificity between plants and the necessary Agrobacterium strain for transformation. As we found out, getting hold of the correct Agrobacterium strain for a plant species (if one is even known) is extremely hard, reaching up to hundreds of Euros and months of shipping time. This is made even worse by the confusing and often conflicting nomenclature of existing strains (De Saeger et al., 2021^[7]).

Therefore, we created a composite part that aims to improve the efficiency and host range of Agrobacterium strains by leveraging the virG transcription factor as a Master-Switch plant transformation. With it, we hope to enable future teams to further extend the garden of plant projects in iGEM.

The molecular machinery of plant transformation

Both the molecular machinery for plant infection and the DNA fragment that is excised and integrated into the plant genome are located in a nonessential, mobile plasmid called the Ti or Ri plasmid (for Agrobacterium tumefaciens and rhizogenes, respectively). In its infection cycle, the metabolites released by plant wounds attract Agrobacterium chemotactically. These same phenolic compounds also activate the virulence response in Agrobacterium.

Figure 1: XXX

The T-DNA is composed of genes for the tumor formation and for the production of metabolites that are used as a carbon source by the bacterium, these are flanked by direct repeats (T-DNA borders) that are recognized by vir genes and are essential for the excision, transport, and integration in the plant genome. The T-DNA border sequence plays a fundamental role in the biotechnology applications of Agrobacterium, as the genes of interest to be transformed in plants must also be flanked by direct repeats (Ozyigit et al.^[11], 2013). Which genes are included in the T-DNA are the main factor in differentiating Agrobacterium strains, while the T-DNA of A. tumefaciens strains promoters the formation of overground tumors, the gene products of A. rhizogenes T-DNA result in the formation of the hairy root phenotype (Bouchez & Tourneur^[4], 1991; Slightom et al.^[12], 1986).

The virulence (vir) region is a cluster of ~30 coding sequences responsible for the DNA transfer mechanisms, two of those genes, VirA and VirG, form a two component system in which VirA is a transmembrane histidin kinase and VirG the cytoplasmic response regulator (Cho & Winans^[5], 2005). Upon binding to plant phenolic compounds (acetosyringone etc.), VirA phosphorylates VirG, which in turn functions as a transcription factor that binds to vir gene promoters and activates their transcription. Therefore, VirG can be seen as a “master regulator” of Agrobacterium virulence.

Controlling the expression of this master regulator through its overexpression or the inclusion of extra copies from more potent strains has been shown to increase transformation efficiency and host range (Anand et al.^[1], 2019).

The virG Master-Switch construct

We propose the use of a helper plasmid that decouples the expression of VirG from the VirA two component system, skipping the need for optimizing the bacteria growth medium and the use of phenolic compounds that emulate the plant response. By fine tuning the expression of this “master-switch” of Agrobacterium virulence, we aim to take control of the transformation machinery and tailor it to individual plant species. Here, we present the result of our engineering efforts and the multiple iterations of the design-build-test-learn cycle that culminated in our part.

Constitutive expression

The initial design for our construct relied on maximizing the expression of a second copy of the endogenous virG from A. rhizogenes Arqua1. This was based on the assumption that a maximum induction of the virulence genes would lead to the best transformation results. This approach was also used to improve transformation efficiency in celery and rice (Liu et al.^[14], 1992). As a backbone, we chose the pSRK L1 entry vector, which was provided by the lab of our PI Anke Becker, has a pBBR1 broad host range ori. Based on the data gathered from our Anderson Promoter characterization, we identified J23102 as the strongest constitutive promoter, and designed a construct that used this promoter to drive the expression of the endogenous VirG CDS basic part we amplified from A. rhizogenes ARqua1.

Figure 2:

However, after more thorough research, and - most importantly - after consulting with Sebastian Concioba, we found that strong virulence induction might be an extremely high metabolic burden for the cell, leading to slower growth and possibly even an overall decrease in transformation efficiency. This prompted us to return to the drawing board and rethink how our composite part could work.

Inducible expression

Next, we decided to change the design by using an inducible promoter system. By doing that, not only could we delay the virulence response until it was actually needed, but also open up the potential for fine-tuning the virulence response for each plant species of interest. Here we faced another challenge, the lack of basic parts that are well characterized in Agrobacterium. While some efforts have been made in shedding light on the function of inducible systems in this organism, its volume still pales in comparison to other model organisms.

We selected 9 promoters from the “Marionette Collection”, which contains a number of inducible systems highly optimized (in E. coli) for high dynamic range and low leakyness (Meyer et al.^[9], 2019). Additionally, Ptrc and Ptau were also included (Mostafavi et al.^[10], 2014; Stukenberg et al.^[13], 2021). Another consideration made when selecting the promoter systems to characterize was to include ones that use non-phenolic compounds as inducers (Ptau, IPTG, Pbetl, and Pbad), in the hope of minimizing cross talk with the native VirA/VirG two component system.

Figure 3:

The results in Fig. 3 show relative luminescence (RLU) output from H2O mock induction and maximum induction. This experiment demonstrated that most promoters did not respond significantly to induction in A. rhizogenes ARqua1, notable exceptions were Ptac, Pvan, PnahR and Ptau. With first two showing the highest overall induction strength and Ptau the widest dynamic range, in fact, the baseline expression of Ptau was as low as the dummy promoter, both at the threshold of detection for the plate reader used in the experiment, this demonstrates that the expression of Ptau is tightly regulated and has virtually zero leakiness. Overall, PnahR appeared to have a good middle ground between expression strength and orthogonality, and was selected for driving the expression of VirG in our Master-Switch construct.

Cross-talk assay

Sequence and Features

Assembly Compatibility:

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References

[1] Anand, A., Che, P., Wu, E., & Jones, T. J. (2019). Novel Ternary Vectors for Efficient Sorghum Transformation. In Z.-Y. Zhao & J. Dahlberg (Eds.), Sorghum (Vol. 1931, pp. 185–196). Springer New York. https://doi.org/10.1007/978-1-4939-9039-9_13

[2] Bahramnejad, B., Naji, M., Bose, R., & Jha, S. (2019). A critical review on use of Agrobacterium rhizogenes and their associated binary vectors for plant transformation. Biotechnology Advances, 37(7), 107405. https://doi.org/10.1016/j.biotechadv.2019.06.004

[3] Barton, I. S., Fuqua, C., & Platt, T. G. (2018). Ecological and evolutionary dynamics of a model facultative pathogen: Agrobacterium and crown gall disease of plants. Environmental Microbiology, 20(1), 16–29. https://doi.org/10.1111/1462-2920.13976

[4] Bouchez, D., & Tourneur, J. (1991). Organization of the agropine synthesis region of the T-DNA of the Ri plasmid from Agrobacterium rhizogenes. Plasmid, 25(1), 27–39. https://doi.org/10.1016/0147-619X(91)90004-G

[5] Cho, H., & Winans, S. C. (2005). VirA and VirG activate the Ti plasmid repABC operon, elevating plasmid copy number in response to wound-released chemical signals. Proceedings of the National Academy of Sciences, 102(41), 14843–14848. https://doi.org/10.1073/pnas.0503458102

[6] Colognori, D., Trinidad, M., & Doudna, J. A. (2023). Precise transcript targeting by CRISPR-Csm complexes. Nature Biotechnology, 1–9. https://doi.org/10.1038/s41587-022-01649-9

[7] De Saeger, J., Park, J., Chung, H. S., Hernalsteens, J.-P., Van Lijsebettens, M., Inzé, D., Van Montagu, M., & Depuydt, S. (2021). Agrobacterium strains and strain improvement: Present and outlook. Biotechnology Advances, 53, 107677. https://doi.org/10.1016/j.biotechadv.2020.107677

[8] Gelvin, S. B. (Ed.). (2018). Agrobacterium Biology: From Basic Science to Biotechnology (Vol. 418). Springer International Publishing. https://doi.org/10.1007/978-3-030-03257-9

[9] Meyer, A. J., Segall-Shapiro, T. H., Glassey, E., Zhang, J., & Voigt, C. A. (2019). Escherichia coli “Marionette” strains with 12 highly optimized small-molecule sensors. Nature Chemical Biology, 15(2), Article 2. https://doi.org/10.1038/s41589-018-0168-3

[10] Mostafavi, M., Lewis, J. C., Saini, T., Bustamante, J. A., Gao, I. T., Tran, T. T., King, S. N., Huang, Z., & Chen, J. C. (2014). Analysis of a taurine-dependent promoter in Sinorhizobium meliloti that offers tight modulation of gene expression. BMC Microbiology, 14, 295. https://doi.org/10.1186/s12866-014-0295-2

[11] Ozyigit, I. I., Dogan, I., & Artam Tarhan, E. (2013). Agrobacterium rhizogenes-Mediated Transformation and Its Biotechnological Applications in Crops. In K. R. Hakeem, P. Ahmad, & M. Ozturk (Eds.), Crop Improvement: New Approaches and Modern Techniques (pp. 1–48). Springer US. https://doi.org/10.1007/978-1-4614-7028-1_1

[12] Slightom, J. L., Durand-Tardif, M., Jouanin, L., & Tepfer, D. (1986). Nucleotide sequence analysis of TL-DNA of Agrobacterium rhizogenes agropine type plasmid. Identification of open reading frames. Journal of Biological Chemistry, 261(1), 108–121. https://doi.org/10.1016/S0021-9258(17)42439-2

[13] Stukenberg, D., Hensel, T., Hoff, J., Daniel, B., Inckemann, R., Tedeschi, J. N., Nousch, F., & Fritz, G. (2021). The Marburg Collection: A Golden Gate DNA Assembly Framework for Synthetic Biology Applications in Vibrio natriegens. ACS Synthetic Biology, 10(8), 1904–1919. https://doi.org/10.1021/acssynbio.1c00126

[14] Liu, C.-N., Li, X.-Q., & Gelvin, S. B. (1992). Multiple copies of virG enhance the transient transformation of celery, carrot and rice tissues by Agrobacterium tumefaciens. In Plant Molecular Biology (Vol. 20, Issue 6, pp. 1071–1087). Springer Science and Business Media LLC. https://doi.org/10.1007/bf00028894